The synthesis of mesoporous molecular materials using pore forming materials or xe2x80x9ctemplatesxe2x80x9d has emerged as an important area of research. However, currently the most successful of such templates for directing the mesophase formation are surfactants. The choice of such templates is relatively limited and many surfactants, e.g. alkylammonium ions and aikylamines, are costly and toxic, particularly with respect to biologically active agents.
The synthesis of mesoporous materials using surfactants as templates has been studied extensively, and the materials were intended for applications in catalysis, biochemical separation technology and molecular engineering. Surfactants, ionic and neutral have been the most commonly used pore forming materials for directing the formation of mesoporosity. The ionic pathways are based on charge matching between the ionic surfactants and ionic inorganic precursors through electrostatic interaction. Neutral surfactants are theorized to use hydrogen bonding between the surfactants and the precursors to direct formation of mesostructures.
The discovery of the M41S family of mesoporous silicate and aluminosilicate molecular mesoporous materials, or sieves, using surfactant templated hydrothermal sol-gel processes was reported in 1992 by C. T. Kresge et al., Nature, xe2x80x9cOrdered Mesoporous Molecular Sieves Synthesized By A Liquid Crystal Template Mechanism,xe2x80x9d vol. 359, page 710 (1992). This discovery drew great interest because of the potential applications of such mesoporous materials as catalysts, catalyst supports, separation media, and host material for inclusion compounds. Numerous mesoporous or nanoporous materials have been synthesized and the pore diameter extended from less than 13 xc3x85 for conventional zeolites to about 100 xc3x85 and lately up to 300 xc3x85. Many synthetic routes and strategies have been developed to yield a wide diversity of materials of various framework chemical compositions and pore structures. The mostly commonly used templates, however, for directing the mesophase formation have been ionic or non-ionic surfactants. A number of mechanistic pathways have been proposed to account for the formation of nanophase structures, based on the electrostatic interactions and charge-matching for the systems with ionic surfactant templates and the hydrogen bonding interactions for the systems with neutral surfactant templates such as amines and polyethylene oxide (PEO) copolymers. A covalent bonding pathway has also been proposed. In addition, mesoporous materials may also be prepared from interlayer crosslinking of a layered silicate through ion exchange reactions with organic cations.
MCM-41 mesoporous materials have an array of hexagonal arrangements of uniform mesopores of 15 to 100 xc3x85 in diameter, which could be controlled by the hydrophobic alkylchain length of ionic surfactants or with the aid of auxiliary organic compounds as spacers. The ionic templates are usually removed by high temperature calcination or ion exchange. Strong electrostatic interactions among the ionic surfactants and the silicate intermediates result in matrices with limited pore wall thicknesses of 0.8-1.3 nm that are influenced little by pH in the synthesis. As a consequence, the materials often have limited thermal stability as evidenced by significant pore contraction or even structure collapse during calcination. Nonetheless, MCM-41 and their analogues have been explored for many applications. They may serve as model adsorbents for the study of gas sorption in mesoporous solids, as catalysts especially when transition metal elements or organic functional groups are incorporated into the framework structure, and as host materials for inclusion of other molecules.
With neutral, primary-amine surfactants as the template, a family of hexagonal mesoporous silicas have been prepared. The pore size may be adjusted by changing the hydrophobic tail length of the amines. The template can be removed by solvent extraction. The mesoporous materials have greater wall thicknesses (1.7-3.0 nm) due to the absence of electrostatic or charge-matching effects, and thus higher thermal stability than M41S materials. However, the materials exhibit both complementary textural and framework-confined mesoporosity. The toxicity of amines also remains a concern if a large scale production is intended.
The use of neutral, polymeric PEO surfactants as pore forming materials has been demonstrated as advantageous in solving the problems of ionic surfactant charge-matching and organic amine toxicity, since the PEO surfactants are neutral, non-toxic and biodegradable. In addition, the pore size can be controlled by varying the size and structure of the PEO surfactant molecules though the channels are largely disordered. Recently, highly ordered porous silicas (20-300 xc3x85) with large wall thickness values of 3.1-6.4 rum and pore volumes up to 2.5 cm3/g were synthesized by using alkyl PEO oligomeric surfactants and poly(alkylene oxide) block copolymers as templates in strongly acidic media, however, such acidic media are not biocompatible and would present problems with respect to applications involving biologically active agents. In addition, it is difficult to remove such templates using solvent extraction due to their high molecular weight. Further, such attempts do not generally form transparent, monolithic materials which are important for specific applications.
One area of application of microporous and mesoporous materials is for the immobilization of biologically active agents within these materials. Immobilization of enzymes, in particular, has been a subject of extensive research efforts because of its immense technological potentials. Among the popular methods of immobilization is formation of chemical bonding between enzymes and a solid support, which often alters the enzymatic activity. A variety of enzymes and other bioactive substances have been entrapped in inorganic oxides such as silica for biocatalysis and biosensor applications through conventional sol-gel processes. However, because of the microporous nature of the conventional silica matrices (i.e. typical pore diameter less than 15 xc3x85 and pore volume less than 0.25 cm3/g), the catalytic activities of enzymes are hindered by low diffusion rates of substrate molecules and poor accessibility of enzymes inside the materials.
Mesoporous materials are valuable to the life sciences because the larger pore size in comparison with microporous materials allows for a more suitable environment and better mass transfer for biologically active agents. Much of the prior art involving the immobilization of biologically active agents in porous materials involves use of microporous materials, not mesoporous materials. Biologically active agents previously have been bound to microporous materials, but the pore diameters result in steric hindrance and mass transfer limitations on the use of such materials in biological reactions.
Recent advances in the development of mesoporous materials have enabled the immobilization of biologically active agents, but these techniques primarily involve the use of surfactants as templating agents. The syntheses are either detrimental to the activity of biologically active agents, or employ extreme synthesis conditions (such as high temperature, or low pH). Further, such procedures generally do not provide transparent, monolithic mesostructured materials having immobilized enzymes have been achieved using such methods.
As such, there is a need in the art for an easy-to-synthesize mesoporous material. There is further a need for a process for making such materials, and in which the materials can be biocompatible. There is also a need for a method for immobilizing biologically active agents which enables biologically active agents to fit in the mesopores while also preserving biological activity. Those prior art methods using templating agents have been generally unsuccessful in that the templating agents used in forming the mesoporous materials were toxic or denaturing to the biologically active agents. There is also a need for a transparent, monolithic, i.e., millimeter sized, mesoporous material as such materials are useful for biosensor applications.
Immobilization of enzymes, and other biologically active agents, by entrapment in a gel matrix is based on the occlusion of an enzyme within a constraining structure tight enough to prevent the relatively large protein molecules from diffusing into the surrounding media, while still allowing penetration of the relatively small substrate and product molecules in and out of the matrix. Due to the advantages in their generality, the methods which are widely used for entrapping biologically active agents, include adsorption on an inert support, encapsulation within a semipermeable membrane, covalent crosslinking of the protein molecules or coupling to a support. The successful immobilization of the enzyme alkaline phosphatase by entrapment in silica sol-gel glasses drew great interest and spawned many researches on the sol-gel immobilization of various bioactive species. Since then, enzymes, whole cells, antibodies and other proteins have been immobilized via the sol-gel processing in various ceramic or glass matrices in the form of fibers, thin films, monoliths or granules for biocatalysts and biosensors applications.
However, the conventional sol-gel process for the formation of ceramic or glass materials consists of hydrolysis of a metal alkoxide precursor, typically tetramethylorthosilicate or tetraethylorthosilicate for forming silica, in the presence of an acid or, less often, a base catalyst, followed by the polycondensation of the inorganic intermediates and evaporation of solvents, giving a porous solid gel. This type of air-dried xerogel typically possesses numerous pores or channels well below 15 xc3x85 in diameter, depending on the synthesis conditions. Such materials are often used as the host matrices for the encapsulation of various types of chemicals, especially of recent interest being biologically active agents, due to the relatively mild synthesis conditions and easy manipulation. In direct immobilization by entrapment, no chemical bonding is necessary between the host matrix and the guest substance, since the target object is physically imprisoned in the host cages with open channels which allow the reactants and products to migrate through but, ideally, not the entrapped guest. This method is especially suitable for the immobilization of bulky biologically active agents in that no chemical modification of the biomolecules is needed for an effective trapping through primary bonding and the preparation process often involves relatively mild conditions. The resultant ceramic or glass matrix provides a chemically and thermally stable and inert environment for the immobilized species. In addition to the advantages of continuous use and easy separation and recovery of the heterogeneous biocatalyst from the reaction mixture, previous work shows that immobilized enzymes have greater thermal and biochemical stability than the free enzymes in solution.
Despite the advantageous properties of these matrices, however, the problem of internal diffusion-controlled mass transport of the substrate and product inside the rigid sol-gel matrix has been remained a key issue due to limited pore or channel size that is hard to bring under control in the one-step direct immobilization processes previously employed. And consequently, this type of immobilization technique has not been generally used in place of traditional methods for protein and cell immobilization.
After immobilized in a matrix, the enzyme is required to retain at least part of its original catalytic activity. The apparent rate of reaction for immobilized enzymes with their substrates in various organic or inorganic matrices is often found to be diffusion-controlled. This is even a more severe problem for the immobilized enzymes in the highly crosslinked rigid sol-gel materials. This problem is intensified due to the limited accessibility of entrapped enzyme and limited rate of internal diffusion of the substrate due to the narrow matrix pores or channels which are not large enough to eliminate mass transport resistances in the as-synthesized biogels. The kinetic studies may often reflect only the apparent reaction rate of immobilized enzyme with its substrate, which is determined by the rate of internal mass transport rather than by the enzyme-catalyzed reaction kinetics, especially for an enzyme of high activity. In this case, the rate of reaction manifests the diffusion-controlled encounter of the enzyme and its substrate. Therefore, the actual or inherent catalytic activity of immobilized enzyme is hard to determine and is very often underestimated, which easily leads to the conclusion that the enzyme is structurally modified or partially denatured or deactivated during or after immobilization. Also, the internal mass transfer resistances severely limit the practical applications of immobilized enzymes for various occasions such as biosensors where short response intervals are crucial or where macroscopic monoliths are needed.
Therefore, there is a need for improved matrices with larger channels or pore diameters to facilitate the migration of the substrates and products through the pores to adapt such materials for practical applications.
Mesoporous sol-gel materials may be an alternative host for the development of improved biogels. Previously, mesoporous silicate and aluminosilicate, as well as other metal oxides with pore diameters in the range 20 to 100 xc3x85, and up to 300 xc3x85 have been synthesized based on the template-directed hydrothermal reactions according to the varied ionic or nonionic surfactant templating pathways. In most cases, the template-based synthesis approaches to the mesostructured materials are difficult to adapt for direct immobilization of biologically active agents due to the severe reaction conditions necessary. Although indirect enzyme immobilization in a mesoporous MCM-41 molecular sieve has been tried, simple, direct immobilization of biologically active agents in such mesoporous sol-gel materials has not yet been achieved.
As discussed above, when an enzyme with high activity is immobilized in a porous matrix, its activity will often be determined by the availability of the substrate in the near vicinity of its surface or active site. The apparent enzymatic reactivity may be limited by the rate of internal diffusion of its substrate in the matrix instead of the kinetics in case of the kinetic reaction rate greater than the diffusion rate. That is, the rate of reaction will be higher if it is not limited by the rate of internal diffusion of the substrate, and sometimes, of the product through the carrier. In the extreme cases, the substrate molecules will never have a chance to encounter the enzyme molecules entrapped in the core of a relatively large particulate because they have already been consumed and converted into the products by the enzyme molecules in the outer shell before they can reach the particle center. This will yield a lowered apparent activity when the calculation is based on the total amount of enzyme entrapped. It is known that volume diffusion proceeds in large pores exceeding 100 nm while Knudsen diffusion proceeds in narrow pores below 100 nm in which the mean free way of the molecules exceeds the pore diameter, and sol-gel materials having pore diameters in the range of micropores ( less than 20 xc3x85) or mesopores (20-500 xc3x85) fall into this category. It has been expected and pointed out that diffusion limited reaction rates have been evident in microporous sol-gel materials and virtually an obstacle in the practical applications.
As such, there is a need in the art for a mesoporous material for use in applications with biologically active agents that can be prepared using a pore forming material that is readily available, non-toxic and inexpensive, and is free from the undesirable effects upon biologically active agents such as denaturation or toxicity. Further, there is a need in the art for a simple, controllable synthesis method of such a mesoporous material, and for immobilizing a biologically active agent within such a mesoporous material in which the biologically active agent can retain an acceptable or high degree of its native activity. In addition, it would be desirable to be able to control the desired pore size and volume of such mesoporous materials for various biologically active agents of different sizes to optimize mass transfer, and to achieve monolithic and transparent materials.
The invention includes a method for making a mesoporous material which comprises forming an aqueous solution having an organometallic compound, adding a solution comprising a pore forming material to form a sol-gel matrix by polycondensation, drying the sol gel matrix and removing the pore forming material from the dried sol-gel matrix to thereby form a mesoporous material. The pore forming material is selected from the group consisting of monomeric polyols, polyacids, polyamines, carbohydrates, oligopeptides, oligonucleic acids, carbonyl functional organic compounds, and mixtures and derivatives thereof. In a preferred embodiment, the pore forming material is a nonsurfactant, polar compound capable of forming hydrogen bonding.
In one embodiment of the above method, a biologically active agent is added after adding the solution of the pore forming material.
The invention further includes a mesoporous material having pores and formed from a sol-gel matrix comprising a pore forming material and having a surface area of at least about 600 m2/g , a pore volume of at least about 0.5 cm3/g and an average pore size diameter of from about 20 xc3x85 to about 100 xc3x85, wherein at least about 50% of pores in the mesoporous material are mesopores.
The invention further includes a method of using a mesoporous material with a biologically active agent which comprises preparing a mesoporous material having pores from a sol-gel matrix which comprises an organometallic compound and a pore forming material selected from the group consisting of monomeric polyols, polyacids, polyamines, carbohydrates, oligopeptides, oligonucleic acids, carbonyl functional compounds, and mixtures and derivatives thereof, immobilizing a biologically active agent within the pores of the mesoporous material and introducing the immobilized active agent into a biological system.
The invention also includes a mesoporous material comprising pores having an average pore diameter of from about 30 xc3x85 to about 60 xc3x85, wherein a plurality of the pores are interconnected within the mesoporous material and a biologically active agent immobilized within the pores.